The present invention is a method to detect the presence of a live virus in a sample, and more particularly, a method to detect the presence of live hepatitis viruses, especially hepatitis B virus, in a body fluid, such as serum, or in a biological preparation, such as a vaccine.
In simplest terms, a vaccine for injection into a mammal for immunization from a disease contains weakened or dead viruses. The injection of the weakened or dead virus into the mammal causes natural antibodies to develop within the mammal and ultimately results in an immunization by that mammal toward the live virus.
It is essential to the proper use of any vaccine that it does not contain any live virus that could cause disease. Of course, since the vaccine is actually made from a live virus which is either weakened or killed, there is a possibility that the vaccine might contain an infectious virus. Injecting an infectious virus into a primate would contribute toward the primate's contracting the particular disease and therefore would not accomplish the objective disease.
Accordingly, a vaccine must be tested to determine whether or not any infectious virus is present. At this time, there is no known way to detect the presence of many infectious viruses, including hepatitis viruses, in a sample except for a very costly and time consuming use of primates. Essentially, the vaccine is injected into a chimpanzee and after waiting a period of time of approximately one year, if the chimpanzee does not develop the disease, the vaccine is considered safe. This process is time consuming, as a sufficient period of time must go by to ensure that the disease does not surface in the animal. The method is expensive as each animal used is expensive and cannot be used more than once.
Besides vaccines, humans receive a variety of blood products such as clotting factors that have been associated with transmitting diseases, such as viral hepatitis. A method to detect the presence of hepatitis viruses in these products would aid in ensuring the safety of these products for human use.
Viral hepatitis B is a very serious illness. A large proportion of the world's population dies from complications of this virus. The virus is transmitted perinatally, sexually, and from a variety of other sources. Because of the danger associated with the disease, there is an incentive to immunize the susceptible populations against this virus much the same way immunization against smallpox or polio has been accomplished.
At present, the chimpanzee infection model is used to detect the presence of live hepatitis B virus ("HBV") in a body fluid or biological preparation. In vitro assays are available, but they are limited to those which measure the presence of viral products, such as DNA and proteins that the virus makes. These assays do not measure the biological viability of the virus. Therefore, even when the virus is dead, these assays would still test positive for HBV because of the presence of its by-product in the sample.
Infection with hepatitis B virus can lead to chronic hepatitis, cirrhosis and primary liver carcinoma. In the past, efforts to devise an in vitro model for hepatitis B virus infection concentrated on liver cells because that is where the disease often expresses itself; the liver appears to be the major organ affected by the virus.
Recently, researchers have broadened their search and HBV DNA has been detected in the kidney, pancreas, spleen, vascular endothelium, skin and semen of infected patients. It has also been shown that circulating mononuclear cells of chronically infected patients contain HBV DNA. Romet-Lemonne et al have found that a small percentage of bone marrow cells from infected patients contained hepatitis B virus surface antigen (HBsAg) and hepatitis B core antigen (HBcAg). See, Romet-Lemonne et al, "hepatitis B virus infection in cultured human lymphoblastoid cells", Science 221: 667-669 (1983). Yet, to this date a reliable in vitro model to determine the presence of live hepatitis B virus has not been developed.
Recently, Young et al demonstrated that in blood of people who are diagnosed as having severe anemia (a disease which expresses itself by noticeable suppression of the growth and differentiation of bone marrow cells), a virus was present which inhibited the growth and differentiation of certain bone marrow stem cells (CFU-E). Young et al developed an in vitro assay that detects the inhibition of growth of the stem cell when exposed to a sample containing the virus to indicate the presence of the virus. See, N. S. Young et al, "Characterization of a Virus that Causes Transient Aplastic Crisis", Journal of Clinical Investigation, 73:224-230, 1984. This assay was not successful, however, to detect the presence of hepatitis B virus in vitro; sera from patients with HBV-associated hepatitis failed to inhibit the growth and differentiation of bone marrow stem cells.
Accordingly, it is an object of the present invention to provide an assay to detect the presence of live virus in vitro.
It is another object of the present invention to provide an economic and accurate assay to detect the presence of live hepatitis viruses in vitro.
Still another object of the present invention is to provide an assay to detect the presence of live hepatitis B virus in a vaccine.